Recent work has revealed an unexpected role for the anionic phospholipid, palmitoyl-oleoyl- phosphatidylglycerol (POPG) as an antagonist of multiple Toll-like receptors, and a potent inhibitor of Respiratory Syncytial Virus (RSV) and Influenza A virus (IAV) infection. The emerging view is that POPG functions as a decoy ligand for multiple Toll-like receptors (TLRs 1,2,3,4 and 6) and competitively blocks their engagement by native ligands present on microbes. Likewise, the phospholipid competitively inhibits recognition of the cell surface attachment ligands for RSV and IAV and thereby prevents the viruses from binding to the plasma membrane and infecting epithelial cells. Currently, the general features of POPG action against TLRs and viruses are clearly understood from in vitro and in vivo studies, but the specific molecular mechanisms of action have not been elucidated. In this proposal we will focus upon the physical interactions of POPG and related phospholipids with Toll-like receptor 3 (TLR3) and the recently emergent pandemic H1N1 (pH1N1) virus. The goals are to define the structural features of phospholipids, TLR3 and the envelope proteins of pH1N1 that are essential for the lipid-protein interactions and the subsequent inhibition of biological effects. To accomplish these goals we will pursue three Specific Aims. In Aim 1 we will examine the elements of lipid structure required to inhibit TLR3 activation and IAV infection by using a library of POPG analogs. In Aim 2, we will identify the phospholipid binding site on TLR3 and determine its relationship to the double stranded RNA binding sites on the molecule. In Aim 3, we will identify the POPG binding site(s) on pH1N1 envelope protein(s) and determine its relationship to the sialic acid binding domain of the H1 protein. From these studies we expect to define specific molecular interactions and mechanisms by which POPG and structurally related molecules suppress IAV infection and the TLR3-dependent inflammatory processes that follow viral infection. This information will provide new pharmacological insights for preventing and treating IAV infections and reducing the pathological consequences of the inflammatory sequelae. There is significant potential for this work to identify novel lipids that will function as antagonists of newly emergent and highly virulent strains of IAV.